PROJECT SUMMARY Acute lymphoblastic leukemia (ALL) and neuroblastoma (NB) are the most common cancers in children. Currently treatment options are often associated with severe side effects. Therefore, there is a strong unmet medical need to develop targeted-drugs with higher therapeutic indexes for improved treatment outcome. The oncoproteins MDM2 and XIAP are important cell-survival proteins in tumors. Elevated MDM2 and XIAP expression is associated with disease progression and poor treatment outcomes. They represent very attractive cancer drug targets. While several MDM2-p53 and XIAP inhibitors exist, none of them made to the market yet. A recent collaboration at UTHSC and Emory led to the discovery and patenting of the small molecule MX69- 52 as a MDM2/XIAP dual inhibitor. MX69-52 binds to the MDM2 RING domain and disrupts its interaction with XIAP IRES, resulting in simultaneous inhibition of both MDM2 and XIAP, leading to cancer cell apoptosis. MX69- 52 has an MTD ?200 mg/kg and is effective against leukemia xenograft models at 15 mg/kg, suggesting a large therapeutic index. SEAK Therapeutics licensed this patented scaffold and aims to develop the most potent isomer within the isomeric mixture MX69-52, namely MX69-52d, as a more effective drug for pediatric cancers. SEAK Therapeutics proposes in this Phase I SBIR to characterize and de-risk MX69-52d as a viable clinical candidate and to provide proof of concept for this approach. Aim 1. Generate sufficient amounts of the most potent optical isomer MX69-52d and determine its absolute structure. Milestones: (1) produce 500 mg of MX69-52d (purity ? 98%); (2) unambiguously determined the absolute structure of MX69-52d to enable future development of a stereo-specific synthesis. Aim 2. Confirm MDM2/XIAP dual inhibition by MX69-52d and evaluate its potential off-target effects against a panel of physiologically important targets. We will confirm that MX69-52d maintains its mode of action similar to that of its isomeric mixture MX69-52. We will also map its potential off-target effects using Cerep?s Safety47 panel. Milestones: (3) confirmed dual MDM2/XIAP inhibitory abilities of MX69-52d in both biochemical and cellular assays; (4) identified potential interactions at 10 M on physiologically important targets (e.g., GPCRs, ion channels, transporters) to further de-risk MX69-52d. Aim 3. Determine maximum tolerated dose (MTD) and pharmacokinetic (PK) parameters of MX69-52d, evaluate its anticancer activities in xenograft models, and assess its potential in vivo toxicity to normal cells/tissues. We will evaluate the improved efficacy and therapeutic index of MX69-52d using two mouse models of pediatric cancers. Milestones: (5) the MTD of MX69-52d (?200 mg/kg) and PK parameters; (6) demonstrated anti-cancer efficacy without acute toxicities at ?20 mg/kg. Success of this work will set the stage for a Phase II SBIR focusing on the development of MX69-52d or an improved analog through pre-IND studies, with the goal to develop a more effective drug for pediatric cancers.